Jag inleder med att berätta om en doktorsavhandling som berör stroke och de konsekvenser själva läkningsprocessen kan orsaka. En intressant iaktagelse få säkert har kunskap om.Vid en stroke utlöses en kedja av händelser,ex så försöker hjärnan själv fixa traumat. Det genom att det centrala nervsystemet får en signal att vakna och skicka celler till det skadade området i syfte att påbörja reparationsarbetet. När det sker passerar dessa celler det som kallas blodhjärnbarriären.Denna barriär är till för att skydda hjärnan mot intrång av allehanda för hjärnan farliga element som bakterier,virus..mm. Men eftersom det skett ett trauma släpper denna barriär igenom hjälptrupperna (friska celler). När det sker exponeras hjärnan vilket är en risk. I denna studie berättar doktoranden att själva hjälptrupperna (friska celler) kan innebära risk för hjärnan.
Först en utvikning om vad en stroke är.
En stroke definieras som konsekvens av någon av 2 händelser.
1. En propp har täppt till flödet av blod i ett blodkärl och hjärnan får inte syre till den blodbanans celler. Dessa börjar då dö och med det funktioner som styrs av området. Ex talet eller en kroppsdel som arm eller ben.
2. En blödning har uppstått och det ansamlas blod i någon av hjärnhalvorna. Trycket som då sker i den skadade hjärnhalvan börjar ligga mot den oskadade hjärnhalvan.Man har en massiv hjärnblödning på gång som utan insats snabbt riskerar att leda till hjärndöd. Tänk er ett slutet system som max innehåller 1 liter vätska.Plötsligt brister ett rör i systemet och det sprutar ut vätska. Vätska som inte får plats.Utan åtgärd sprängs systemets väggar så småningom.
1. En propp har täppt till flödet av blod i ett blodkärl och hjärnan får inte syre till den blodbanans celler. Dessa börjar då dö och med det funktioner som styrs av området. Ex talet eller en kroppsdel som arm eller ben.
2. En blödning har uppstått och det ansamlas blod i någon av hjärnhalvorna. Trycket som då sker i den skadade hjärnhalvan börjar ligga mot den oskadade hjärnhalvan.Man har en massiv hjärnblödning på gång som utan insats snabbt riskerar att leda till hjärndöd. Tänk er ett slutet system som max innehåller 1 liter vätska.Plötsligt brister ett rör i systemet och det sprutar ut vätska. Vätska som inte får plats.Utan åtgärd sprängs systemets väggar så småningom.
För att åtgärda dessa 2 händelser görs följande:
I case 1 sätter läkare in blodförtunnande medel som förhoppningsvis löser upp proppen. Fungerar inte det återstår att gå in i venen och "dra" ut proppen mekaniskt.Samtidigt sätter man in ett s.k "hönsnät" som håller uppe venens väggar som kanske säckat ihop och därigenom skapat proppen.
I case 2 följer läkarna utvecklingen och ser de att trycket blir livshotande öppnar de upp skallbenet och på så sätt minskar trycket samtidigt som de får stopp på själva blödningen. Det ingreppet är förenat med stor risk att skada hjärnan via de omslutande skyddande hinnorna man måste skära upp.Konsekvens får ni lista ut själva.
Men tillbaka till studien.Den är sanslöst omfattande med 176 sidor.Oerhört intressant och ger ny kunskap till en enkel lekman som undertecknad är.
Innan något av casen 1 och 2 har tagits itu med av läkare har kroppen satt igång sin egen hjälpaktion.
Centrala nervsystemet har utlöst signalen att skicka in hjälptrupper med celler utan urskilling,det råder panik. Dessa hjälptrupper kan innehålla celler som egentligen inte har i hjärnan att göra,men som sagt det råder panik och kroppen skickar allt den har för att rädda det viktigaste organet, hjärnan.
Innan något av casen 1 och 2 har tagits itu med av läkare har kroppen satt igång sin egen hjälpaktion.
Centrala nervsystemet har utlöst signalen att skicka in hjälptrupper med celler utan urskilling,det råder panik. Dessa hjälptrupper kan innehålla celler som egentligen inte har i hjärnan att göra,men som sagt det råder panik och kroppen skickar allt den har för att rädda det viktigaste organet, hjärnan.
Studiens författare kallar denna hjälpaktion för ett dubbeleggat svärd.
I olyckliga fall förvärrar denna hjälpaktion hjärnskadan.Det då blodhjärnbarriären forcerats av "fel" celler och skapat ny/nya eländen för den drabbade. Ni får läsa vad och på vilket sätt själva.
Investigating the effects of inflammatory mediators on the neuroinflammation response
Abstract
Ischemic stroke is a devastating event which further stimulates a cascade of inflammatory events in the brain, known as neuroinflammation to occur which is important for repair.
However, this neuroinflammation is a double-edged sword that can also have a detrimental impact within the brain.
Numerous inflammatory markers are known to be stimulated following an ischemic event and one of interest for this study is C reactive protein (CRP) and its isoforms monomeric c reactive protein (mCRP) and native c reactive protein (nCRP).
Several different cell types were used throughout this work which included U937 monocytes which were also differentiated into M0 macrophages.
However, this neuroinflammation is a double-edged sword that can also have a detrimental impact within the brain.
Numerous inflammatory markers are known to be stimulated following an ischemic event and one of interest for this study is C reactive protein (CRP) and its isoforms monomeric c reactive protein (mCRP) and native c reactive protein (nCRP).
Several different cell types were used throughout this work which included U937 monocytes which were also differentiated into M0 macrophages.
Finally, human microglia cell line HMC-3 cell were also used. Lipopolysaccharide (LPS) was used as a positive inflammatory mediator through this work. Pro-inflammatory effects were observed when different cell types were treated with mCRP or nCRP to test for any inflammatory effects.
The liver x receptor agonist (GW3965) was selected for its anti-inflammatory properties and current promising research as a therapeutic in ischemic stroke.
Micro-fragment adipose tissue (MFAT) conditioned media was used to test its potential anti-inflammatory effect.
Levels of inflammatory activity were measured by several different methods including enzyme-linked immunosorbent assay (ELISA), immunofluorescent staining, aggregation analysis and qPCR.
The liver x receptor agonist (GW3965) was selected for its anti-inflammatory properties and current promising research as a therapeutic in ischemic stroke.
Micro-fragment adipose tissue (MFAT) conditioned media was used to test its potential anti-inflammatory effect.
Levels of inflammatory activity were measured by several different methods including enzyme-linked immunosorbent assay (ELISA), immunofluorescent staining, aggregation analysis and qPCR.
Results from this study showed that mCRP can alter the morphology and physiological function of both U937 monocytes and HMC-3 microglia cells.
Increased expression of important pro-inflammatory cytokines was observed, including interleukin 1 beta (IL-1b), interleukin 6 (IL-6), tumour necrosis factor alpha (TNFa), highlighting mCRP pro-inflammatory activity.
The LXR agonist (GW3965) significantly reduced keyproinflammatory proteins after HMC-3 microglia cells were stimulated with either LPS or mCRP inflammatory mediators. However, no up regulation or down regulation was observed in gene expression at any time point after treatment.
Increased expression of important pro-inflammatory cytokines was observed, including interleukin 1 beta (IL-1b), interleukin 6 (IL-6), tumour necrosis factor alpha (TNFa), highlighting mCRP pro-inflammatory activity.
The LXR agonist (GW3965) significantly reduced keyproinflammatory proteins after HMC-3 microglia cells were stimulated with either LPS or mCRP inflammatory mediators. However, no up regulation or down regulation was observed in gene expression at any time point after treatment.
This study demonstrated that mCRP acts as a proinflammatory mediator on HMC-3 and U937 monocytes.
MFAT, was also seen to have key antiinflammatory properties with secreting key cytokines and chemokines. An in vitro inflammatory cell model was used to determine MFAT condition medium can attenuate inflammatory cytokine up regulation.
MFAT attenuated the up regulation of protein and gene expression levels of key cytokines (TNFa, IL-1b, IL-6, and IL-10) which are involved in neuroinflammation after an ischemic event such as stroke.
MFAT, was also seen to have key antiinflammatory properties with secreting key cytokines and chemokines. An in vitro inflammatory cell model was used to determine MFAT condition medium can attenuate inflammatory cytokine up regulation.
MFAT attenuated the up regulation of protein and gene expression levels of key cytokines (TNFa, IL-1b, IL-6, and IL-10) which are involved in neuroinflammation after an ischemic event such as stroke.
In conclusion, this study demonstrated that mCRP could cause morphological and behaviour changes in either U937 monocytes or HMC-3 microglia 6 cells.
Further to this mCRP demonstrated pro-inflammatory activity, whilst the use of GW3965 inhibited its pro- inflammatory activity, therefore reducing TNF-α, IL-6, IL-1b, MCP-1, RANTES.
On another note, MFAT secreted key cytokines and chemokines into conditioned media (IL-1ra, Il-1b, IL-4).
Further to this mCRP demonstrated pro-inflammatory activity, whilst the use of GW3965 inhibited its pro- inflammatory activity, therefore reducing TNF-α, IL-6, IL-1b, MCP-1, RANTES.
On another note, MFAT secreted key cytokines and chemokines into conditioned media (IL-1ra, Il-1b, IL-4).
Materials and Methods (urval)
2.2.3.3 Holomonitor live cell tracking of adherent cells.
HMC-3 microglia cells were seeded at 5% confluence in a 24 well ibidi plate (10,000 cells) in
complete media and left for 24 hours to adhere to the plate surface.
The Holomonitor is set up in a humified chamber with 5 % CO2 at 37°C.
The cells were treated with complete media, LPS 10 ng/ml, mCRP 100 µg/ml and nCRP 100 µg/ml. The plate is sealed with sterilized HoloLids designed for live cell microscope.
Holographic imaging was set up according to manufacturer’s manual, and a minimum of three fields of view per well was selected with image taken every hour for 24 hours.
Parameters that will be measured include cell tracking, cell morphology and cytotoxicity analysis.
The Holomonitor is set up in a humified chamber with 5 % CO2 at 37°C.
The cells were treated with complete media, LPS 10 ng/ml, mCRP 100 µg/ml and nCRP 100 µg/ml. The plate is sealed with sterilized HoloLids designed for live cell microscope.
Holographic imaging was set up according to manufacturer’s manual, and a minimum of three fields of view per well was selected with image taken every hour for 24 hours.
Parameters that will be measured include cell tracking, cell morphology and cytotoxicity analysis.
 |
| Figure 30 - Observing HMC-3 morphological changes caused by mCRP. The effects of mCRP (100 µg/ml) on HMC – 3 cells were investigated. 4 treatments were investigated in total 1) Control (complete media), 2) LPS 10 ng/mL positive control, 3) mCRP and 4 nCRP. 1A-C - Control (3,6 and 24 hrs), B1-3 represents LPS 10 ng/ml. C1-3 represents mCRP 100 μg/ml. D1-3 represent nCRP 100 μg/ml. Images taken on live cell holographic imager at 20x with 3 fields of view per condition and repeated n=3. |
4.3.2 Holographic quantifiable cell morphological and proliferative features after treatment
with CRP isoforms
The effects of mCRP on microglia cells (HMC-3) were analysed using the phase shift holographic
live cell imager. This microscope and the integrated analysis software allowed several different
types of analyses to be completed.
 |
| Figure 32 - Holographic imaging of cells reveal the impact of culture extracellular environment on cell migration and cell motility. HMC-3 single cell tracking analysis performed with the integrated software from the phase shift holographic images. HMC-3 cells were grown and treated with 1) Control, 2) LPS 10 ng/mL positive control, 3) mCRP and 4 nCRP as described in chapter 2 (2.2.3.3). A-D) Single cell 2D movement trajectories from 50 cells chosen at random per field of view were displayed in a rose plot. Original units for cell movement parameters are displayed on the x and Y axis by means of logarithmic scale. E) Migration directness, G) cell motility and H) speed. Cell was tracked over a 24-hour period and quantified from single cell tracking data in which 50 random cells were chosen. Graphs represent Mean ± SEM (two – way ANOVA multi-comparison (turkey)) (p* =< 0.05, p**** <0.0001. 3 fields of view per a well were measured and repeated n=3. |
4.3.3 Determine the kinetic dose response for GW3965.
The kinetic dose response for GW3965 was performed using the live cell imager (holo-monitor)
with the integrated analysis software. An in-depth analysis can be performed on the single cell
tracking cell morphology and further looking into cell count, confluence, and mean cell volume
to determine the effects that the drug compound is having upon the cells.
4.3.4 Determine the kinetic dose response for GSK4112.
Another potential compound was analysed, using the kinetic analysis of the holo-monitor, and
that was GSK4112. A range of doses was selected based on the literature search and a kinetic
dose-response assay was performed to determine if there are any adverse effects of GSK4112
on microglial (HMC-3) cells. Several measurement outputs were analysed during this
experiment, which included cell count, confluence, and mean volume.
Holomonitor microscope system was used to observe and analysis the effects of CRP isoforms
(mCRP and nCRP) on HMC-3 microglia cells. Microglia cells can change migration when
stimulated by inflammation. It is well established that different receptors have different effects
on the migration of microglia cells.
Doktorranden berättar i studien hur man kan överkomma dessa konsekvenser hjälptrupperna i värsta fall orsakar. Nämligen med....*tadaaa* Regenerativ Medicin (stamcellsteknologi). Hon har till och med identifierat var i kroppen man kan ta dessa stamceller för att odla fram bot mot hjälptrupperna.
1.4.1.1 Mesenchymal Stem Cells and their potential in neurological regenerative medicine.
Developing new regenerative therapies to repair the brain after ischaemic stroke is vitally important and one promising candidate is mesenchymal stem cells (MSCs) which are widely investigated as a cell therapy for ischemic stroke (Zheng et al., 2018).
MSCs have antiinflammatory potential and are immune regulatory with immunosuppressive capabilities (Y. Han et al., 2019).
MSCs express several different key surface markers which include CD73, CD90, CD105 as positive and negative marker for CD14, CD34, CD45 and HLA-DR (Camilleri et al., 2016).
MSCs are multipotent stem cells and can be known under different names which include but not limited to mesenchymal stem cells, mesenchymal stromal cells, and mesenchymal progenitor cells (Pittenger et al., 2019).
MSCs can differentiate into all end stage lineages (figure 11) (Musial-Wysocka et al., 2019).
MSCs express several different key surface markers which include CD73, CD90, CD105 as positive and negative marker for CD14, CD34, CD45 and HLA-DR (Camilleri et al., 2016).
MSCs are multipotent stem cells and can be known under different names which include but not limited to mesenchymal stem cells, mesenchymal stromal cells, and mesenchymal progenitor cells (Pittenger et al., 2019).
MSCs can differentiate into all end stage lineages (figure 11) (Musial-Wysocka et al., 2019).
Table 2 is a representation of some of the most researched MSCs with common characteristics and specific cell surface markers.
There are great advantages of using MSCs over other stem cells, however their clinical applications are hindered by research barriers. One example is being able to obtains adequate cell numbers without losing potency during subculture and at high passage numbers (Ullah et al., 2015).
There have been several studies and clinical trials which have conducted safety tests on mesenchymal stem cell-based therapies (Galderisi et al., 2022).
Clinical trials have been conducted and show that MSC therapy for the potential treatment in ischemic stroke is feasible and safe (H. Chen and Zhou, 2022).
There have been several studies and clinical trials which have conducted safety tests on mesenchymal stem cell-based therapies (Galderisi et al., 2022).
Clinical trials have been conducted and show that MSC therapy for the potential treatment in ischemic stroke is feasible and safe (H. Chen and Zhou, 2022).
 |
| Figure 11 - Location MSC can be harvested. MSC can be harvested from several different areas within the human body. |
Min kommentar
Det är svårt att inte bli imponerad av denna doktorrands studier.Hon har tagit sig an ett otroligt komplicerat område och levererar svar läkar och forskarkåren förmodligen kommer ha stor nytta av.
Det är svårt att inte bli imponerad av denna doktorrands studier.Hon har tagit sig an ett otroligt komplicerat område och levererar svar läkar och forskarkåren förmodligen kommer ha stor nytta av.
För att inte nämna alla de som drabbas av komplikationer efter en stroke. Extra "kul" är det förstås att hon listat ut att HoloMonitor är användbar för denna typ av studier.Instrumentet har använts flitigt kan vi se. Och ett plus i kanten får hon för att ta med lösningen på efter-stroke konsekvenser, Regenerativ Medicin via stamcellsteknologin. Undrar om hon visste hur väl lierat den är med HoloMonitor.
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Den andra studien är även den mastig och innehållsrik.Med tanke på ovanstående studies tid för bearbetning och säkert även ork för läsare att ta del av nöjer jag mig med att enbart nämna titel och abstract på tvåan.
Ni får traggla er igenom studien och hitta forskarnas användande av HoloMonitor. 😎
Ni får traggla er igenom studien och hitta forskarnas användande av HoloMonitor. 😎
Ledtråd. Ligger under rubrikerna
2.3. Cell elasticity measurements
2.6. Digital holography
Patient's dermal fibroblasts as disease markers for visceral myopathy
Visceral myopathy (VSCM) is a rare genetic disease, orphan of pharmacological therapy.
VSCM diagnosis is not always straightforward due to symptomatology similarities with mitochondrial or neuronal forms of intestinal pseudo-obstruction.
VSCM diagnosis is not always straightforward due to symptomatology similarities with mitochondrial or neuronal forms of intestinal pseudo-obstruction.
The most prevalent form of VSCM is associates with variants in the gene ACTG2, encoding the protein gamma-2 actin. Overall, VSCM is a mechano-biological disorder, in which different genetic variants
lead to similar alterations to the contractile phenotype of enteric smooth muscles, resulting in the emergence of life-threatening symptoms.
In this work we analyzed the morpho-mechanical phenotype of human dermal fibroblasts from patients affected with VSCM, demonstrating that they retain a clear signature of the disease when compared with different controls.
We evaluated several biophysical traits of fibroblasts, and we show that a measure of cellular traction forces can be used as a non-specific biomarker of the disease.
We propose that a simple assay based on traction forces could be designed to provide a valuable support for clinical decision or preclinical research.
lead to similar alterations to the contractile phenotype of enteric smooth muscles, resulting in the emergence of life-threatening symptoms.
In this work we analyzed the morpho-mechanical phenotype of human dermal fibroblasts from patients affected with VSCM, demonstrating that they retain a clear signature of the disease when compared with different controls.
We evaluated several biophysical traits of fibroblasts, and we show that a measure of cellular traction forces can be used as a non-specific biomarker of the disease.
We propose that a simple assay based on traction forces could be designed to provide a valuable support for clinical decision or preclinical research.
Edit.
Sickna snåla lirare. De har låst in studien idag. Jag har alltså en cache kopia av den som funkar att läsa,men inte att länka. Hursom,här kommer några screen shots istället.
Mvh the99
Som service till alla ev HoloMonitornyfikna forskare : HoloMonitor Demo
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